The goal of this project is to characterize the structure, function, and regulation of the reduced folate carrier (RFC), the major membrane transport system for reduced folate cofactors in mammalian cells. An adequate supply of folates is essential for growth and development of all tissues and there is increasing evidence that folate deficiency contributes to chromosomal instability and malignant transformation. RFC transport is also critical to antitumor activities of methotrexate and a new generation of antifolates typified by Tomudex and Pemetrexed, and RFC alterations result in decreased drug uptake and contribute to antifolate resistance. This renewal application expands on recent significant advances by our laboratory including: (a) characterization of the human RFC (hRFC) gene and demonstration of a remarkable complexity of transcriptional and posttranscriptional controls involving 6 alternately spliced 5'non-coding exons (A1/2,A,B,C,D,E) and unique promoters mapping over 35 kb upstream from the translational start; (b) identification of functionally or structurally important charged amino acids (D88,R133,R373,K411) or domains [transmembrane domain (TMD) 6/7 linker; residues 204-214]; (c) development of a functional "cysteine-less" hRFC and localization of transport function to an exofacial loop domain flanking TMD 1 by substituted cysteine accessibility methods (SCAM) with thiol reactive agents; (d) partial determination of hRFC membrane topology (TMDsl-8); and (e) purification of epitope (His10)-tagged hRFC. For our continued studies, we will in Aim 1 (i) establish the topology of TMDs 9-12 and TMD-loop boundaries, and (ii) map putative binding sites for (anti)folate substrates by SCAM, expression of mouse-human chimera RFCs, radioaffmity labeling of His10-tagged hRFC protein, and site-directed mutagenesis. (iii) hRFC oligomeric structures will be characterized by co-fractionation of HA-/His10-tagged hRFCs, analytical gel filtration, and proteoliposome reconstitution. In Aim 2, we will establish (iv) the patterns of 5'UTR and promoter utilization in human tissues, tumors, and cell lines by real-time PCR and RNAse protection. Other studies will characterize: (v) the major regulatory features (e.g., cis elements, transcription factors) of the hRFC-A1/2, -D, and -E promoters; (vi) posttranscriptional controls including effects of the major 5' untranslated regions (UTRs) and splice forms on translational efficiencies and transcript stabilities, and on hRFC levels and function; and (vii) the significance of novel hRFC isoforms translated from in-frame AUGs in the A1/2 and A 5'UTRs. Based on these results, we will (viii) characterize the transcriptional and posttranscriptional regulation of hRFC by cellular folates and/or by pharmacologic manipulations that alter folate/nucleotide pools. Our studies should identify on the major regulatory properties of the hRFC gene and heterogeneous hRFC transcripts in human tumors and tissues, and the molecular determinants of folate and antifolate substrate binding and membrane translocation by the hRFC protein.